Use of plant cell membrane treatment of obesity

ABSTRACT

The present invention relates to the use of a composition comprising at least one cell membrane fraction or parts thereof, for the reduction of lipolytic activity and/or to retard fat digestion, suppress appetite, body weight and/or lower blood lipids. The invention also relates to the use of said hydrophobic peptide in a pharmaceutical as well as a food composition and methods of treating a mammal with said composition.

This application is a Divisional of U.S. Ser. No. 13/033,871, filed 24Feb. 2011, which is a Divisional of U.S. Ser. No. 11/916,945, filed 24Apr. 2009, which is a National Stage Application of PCT/SE2006/000676,filed 9 Jun. 2006, which claims benefit of Serial No. 0501336-2, filed10 Jun. 2005 in Sweden and which applications are incorporated herein byreference. To the extent appropriate, a claim of priority is made toeach of the above disclosed applications.

FIELD OF INVENTION

The present invention relates to the use of a composition comprising atleast one cell membrane or parts thereof, for the reduction of lipolyticactivity and/or to retard fat digestion, suppress appetite, body weightand/or lower blood lipids. The invention also relates to the use of saidhydrophobic peptide in a pharmaceutical as well as a food compositionand methods of treating a mammal with said composition.

BACKGROUND OF INVENTION

Overweight and obesity has become an increasing world-wide problem.Obesity leads to a concomitant increase in several diseases such asdiabetes, arteriosclerosis, hypertension as well as certain cancerforms. High-fat diet, either alone or added with sucrose, is one of themost important factors causing obesity, since these diets easily promoteovereating. It is therefore of great importance to optimize the controlof appetite for dietary fat to reduce obesity. Satiety for fat is mainlydriven from the intestine, as demonstrated through the infusion of fatinto the intestine, which reduces food intake (Greenberg D. and Smith,G. P., Psychosomatic medicine 58: 559-569, 1996). The reason for thesuppression of food intake under these conditions is the release ofvarious satiety peptides in the intestine by the contact of fat with theintestinal mucosa. Since fatty acids, the products of dietary fat, areabsorbed immediately after their production, a reduced rate of fatdigestion would theoretically optimize the satiety for fat.

The key enzyme during intestinal fat digestion is pancreatic lipase. Theuse of lipase inhibitor (Xenical) as a drug against obesity is wellestablished (Sjöstrom L. et al, Lancet 352: 167-172, 1998). The lipaseinhibitor not only reduces body weight but also improves insulinresistance. Such findings hence provide strong evidence for a role ofintestinal fat digestion on satiety for fat and insulin sensitivity. Thedrawback with this lipase inhibitor is that it inhibits all types oflipases and produces steatorrea due to a strongly impaired fatdigestion. It is therefore of utmost importance to develop a naturalcompound that retards fat digestion in a milder way without causingsteatorrea as side effect. We have found a natural compound of highnutritive value that retards fat digestion, suppresses appetite byincreasing satiety hormones and decreases serum triglyceride levels.

SUMMARY OF THE INVENTION

The present invention relates to the use of a composition, for thereduction of lipolytic activity and/or to retard fat digestion, suppressappetite, body weight and/or lower blood lipids. By reducing thelipolytic activity fat digestion will be retarded and the appetitesuppressed and thereby an enhanced satiety for prevention of for exampleobesity.

In a first aspect the invention relates to the use of a compositioncomprising at least one cell membrane or parts thereof, for thereduction of lipolytic activity and/or to retard fat digestion, suppressappetite, body weight and/or lower blood lipids.

According to a second aspect the invention relates to the use of saidcomposition as a pharmaceutical composition.

According to a third aspect the invention relates to the use of saidcomposition as a food composition.

The invented composition may be used to regulate the appetite, such asfor the treatment of the metabolic syndrome either as a disease or adisorder.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the inhibition of pancreatic lipase by biological membranesfrom leaves. Chloroplast membranes from spinach (filled circles); clover(black squares); Arabidopsis thaliana (triangles); rape (circles) andsugar beet (crosses).

FIG. 2 shows the inhibition of pancreatic lipase by biologicalmembranes: Mitochondria from potato tuber (squares), mitochondria fromchicken heart (filled circles), plasma membrane from spinach leaf(diamonds), membranes from Synechocystis (crosses) and chromatophores ofRhodospirillum rubrum (triangles).

FIG. 3 shows the inhibition of pancreatic lipase by isolated membraneproteins. Light harvesting chlorophyll a/b complex, LHC II (triangles),synthetic polypeptide with the same amino acid sequence (i.e.VIHCRWAMLGALGCVFPELL) as one of the alpha helices of LHCII (crosses),transhydrogenase (diamonds) and cytochrome b₆f complex (squares).

FIG. 4 shows the effect of treatment with chloroplast membranes,(thylakoids) during high-fat diet for eleven days in Sprague-Dawley rat.Food intake with chloroplast membranes (squares) and without(triangles). The daily food intake is given as means±SEM from eightanimals in each group (n=8).

Data on body weight gain, serum triglycerides and plasma cholecystokininafter onset of thylakoid treatment are shown in Table 1.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the context of the present application and invention, the followingdefinitions apply:

The term “cell membrane” is intended to mean a modified or unmodifiednatural or synthetically made biological cell membrane, of animal,plant, or microbial origin, wherein the cell membrane comprises intactcell membranes or fractions thereof as well as parts thereof or mixturesof parts and intact cell membranes, such as the hydrophobic peptides orhydrophobic proteins of said cell membrane. Part of the cell membranemay be between 0.1 to 0.5 μm and may solely comprise one or moremembrane spanning peptides.

The term “hydrophobic peptide” is intended to mean a peptide having atleast 85% hydrophobic amino acid residues selected from the groupconsisting of alanine, valine, leucine, isoleucine, proline,phenylanaline, tryptophane, methionine, glycine, cysteine together witha few amino acids with charged residues such as arginine and glutamicacid.

The term “lipolytic activity” is intended to mean the rate of hydrolysisof lipids by lipases.

The term “membrane spanning peptide” is intended to mean at least theamino acid residues, which form the membrane spanning part of theprotein. The peptide may be one or more membrane spanning parts of onemembrane spanning proteins, such as a stretch comprising between 15 to25 amino acid residues and multiples thereof.

Composition

The invention relates to the use of a composition comprising at leastone cell membrane or parts thereof, for the reduction of lipolyticactivity and/or to retard fat digestion, suppress appetite, body weightand/or lower blood lipids. By reducing the lipolytic activity it ispossible to retard fat digestion, suppress appetite, body weight and/orlower blood lipids. Thereby it is possible for the first time toefficiently regulate the appetite of a mammal, such as a human being oranother animal. By enabling the possibility to regulate the lipolyticactivity a slow formation of fatty acids in the intestine will promoteand prolong satiety.

The composition may comprise at least the membrane spanning part of abiological protein, wherein said membrane spanning part compriseshydrophobic amino acid residues. Accordingly the composition comprises abiological membrane or parts thereof, wherein said biological membranecomprises at least said hydrophobic peptide. The composition maycomprises at least one cell membrane hydrophobic peptide having fromabout 15 to about 25 amino acid residues, such as 2, 3, 4 or 5hydrophobic peptides being derived from one and the same protein ordifferent proteins and the composition may comprise

Biological cell membranes for example occur in all living cells andconstitute a large part of the cell mass. Examples of cell membranefractions according to the invention are cell membrane fractionsobtained from animals, plants, algaes, microorganisms or cell membranefractions of parts thereof, which are synthetic or a mixture thereof. Inprokaryotes, there are single or double plasma membranes and inphotosynthesising bacteria also the photosynthetic membrane, thethylakoids. In eukaryotes the membranes include the plasma membrane, theendoplasmic reticulum, the Golgi membrane, the nuclear membrane, thelysosomal membrane, the mitochondrial membranes and for the green algaeand plants also the chloroplast membranes which include the two envelopemembranes and the photosynthetic membrane, the thylakoids. Biologicalmembranes are composed of proteins and lipids. All biological membranescontain intrinsic membrane proteins with one or several membranespanning polypeptide chains composed of hydrophobic amino acids. Most ofthe lipids, such as phospholipids and galactolipids, form bilayers inwhich the intrinsic membrane/membrane spanning proteins are embedded. Inaddition, extrinsic proteins are attached to the surface of themembrane. The thylakoids are responsible for photosynthesis in plants,green algae, and in the photosynthetic bacteria such as blue-green andpurple bacteria. The thylakoid membrane consists of proteins and lipidsin about 70/30 percent ratio. There are more than 100 different proteinsin the membrane; the lipid fraction is dominated by galactolipids withthe main fatty acids being of the omega-3 type. In addition thethylakoid membrane contains several different pigments, chlorophyll a,chlorophyll b, plastoquinones, the carotenoids β-carotene, luteine,violaxanthin and neoxanthin. This means that the thylakoids have acomposition of high nutrition value and the same applies to syntheticmembranes having the same or substantially the same composition as thethylakoids, i.e., chloroplasts as well as the thylakoids may be used inthe food additive as well as in the food product of the invention.Examples of biological membranes are the chloroplasts or the thylakoidmembranes and the membranes may be obtained from clover, rape, sugarbeet, dandelion, Arabidopsis thaliana, maize, tobacco, sun flower,salad, Chenopodium, Atriplex, spinach and grasses or a mixture thereof.

Accordingly, if the composition comprises at least one hydrophobicpeptide, said peptide may have a length of 16, 17, 18, 19, 20, 21, 22,23 or 24 amino acid residues. Examples of peptides are shown in SEQ IDNO:1, SEQ ID NO:2 or SEQ ID NO:3 or mixtures thereof as well as thepeptides may be operably linked to each other. The amino acid residuesmay be selected from the group consisting of alanine, valine, leucine,isoleucine, proline, phenylanaline, tryptophane, methionine, glycine,cysteine together with a few amino acids with charged residues such asarginine and glutamic acid. The amino acid residues and the peptide maybe synthetic or naturally occurring and the same applies for the aminoacid residues, i.e., they may be natural or synthetic ones as long asthey are hydrophobic or carries a charged residue. The membrane fractionmay be a cell membrane fraction, which has been treated with one or moreenzymes to provide smaller pieces of the cell membranes.

Said biological membrane or part thereof of the invention may have asize distribution of 0.1 μm to about 5 μm, such as 0.1, 0.2, 0.3, 0.4,0.5 or 1.0 μm.

Additionally the hydrophobic peptide may have from about 1 to about 50additional amino acid residues and may also be modified by amidation,esterification, acylation, acetylation, PEGylation or alkylation.

The photosynthetic membranes are the most abundant, with respect tomass, of all biological membranes on earth. Green leaves from plantsconstitute a convenient and abundant source for isolation andpreparation in large quantity of chloroplast membranes for the purposeof this invention.

Biological membranes can be isolated in many different ways, such asthose mentioned in the examples. The most common being to firstdisintegrate the cells mechanically which yields membrane vesicles withdifferent size and composition. Large cell debris are removed by lowspeed centrifugation and the membrane vesicles of the supernatant, then,collected by differential centrifugation or gradient centrifugation.Alternatively the large cell debris is removed by filtration, and themembrane vesicles collected by centrifugation.

It is also possible to design methods, which do not involvecentrifugation. In this case the large cell debris is first removed byfiltration. The cell membranes can then be flocculated i.e. precipitatedby different procedures such as:

-   1 Addition of acid or a base such that a pH is reached when    precipitation is achieved,-   2. Additions of polymers with more or less different charged groups,    which induce precipitation,-   3. Heating the vesicle suspension such as between 40-100 degree    Celsius.-   4. Collection of vesicles by partitioning in an aqueous two phase    system whereby the vesicles are concentrated into a small volume    phase (Albertsson P. A. Partition of cell Particles and    macromolecules. Wiley, New York, 1986)-   5. Collection at an interface of a liquid-liquid two-phase system,    such as the phase system given in 4) or a water-oil phase system.-   6. Collection at an interface of a water-oil phase system leading to    an emulsion.-   7. Adsorption onto solid material such as calcium phosphate, silica,    various ion exchange resins.-   8. Freezing and thawing whereby the water crystals formed    concentrate the vesicles into aggregates, which after thawing    flocculate.

The hydrophobic peptides may be synthesised by standard chemicalmethods, including synthesis by automated procedure. In general, peptideanalogues are synthesised based on the standard solid-phase Fmocprotection strategy with HATU(N-[DIMETHYLAMINO-1H-1.2.3.-TRIAZOLO[4,5-B]PYRIDIN-1-YLMETHYLELE]-N-METHYLMETHANAMINIUMHEXAFLUOROPHOSPHATE N-OXIDE) as the coupling agent or other couplingagents such as HOAt-1-HYDROXY-7-AZABENZOTRIAZOLE. The peptide is cleavedfrom the solid-phase resin with trifluoroacetic acid containingappropriate scavengers, which also deprotects side chain functionalgroups. Crude peptide is further purified using preparativereversed-phase chromatography. Other purification methods, such aspartition chromatography, gel filtration, gel electrophoresis, orion-exchange chromatography may be used. Other synthesis techniques,known in the art, such as the tBoc protection strategy, or use ofdifferent coupling reagents or the like can be employed to produceequivalent peptides.

Peptides may alternatively be synthesised by recombinant production (seee.g., U.S. Pat. No. 5,593,866). A variety of host systems are suitablefor production of the peptide analogues, including bacteria, such as E.coli, yeast, such as Saccharomyces cerevisiae or pichia, insects, suchas Sf9, and mammalian cells, such as CHO or COS-7. There are manyexpression vectors available to be used for each of the hosts and theinvention is not limited to any of them as long as the vector and hostis able to produce the antimicrobial peptide. Vectors and procedures forcloning and expression in E. coli can be found in for example Sambrooket al. (Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1987) and Ausubel et al.(Current Protocols in Molecular Biology, Greene Publishing Co., 1995).

The invented composition may be in any form, such as a natural extractobtained by a conventional method such as one of those mentioned below,as well as being dried, freezed or freeze dried.

The invented composition may be used as a food additive and may beadmixed with other components such as fat, butter, margarine, oils,cream, milk, cheese, brie, flour, juices, soft drinks, teas either priorto being added to a food product or during the addition to the foodproduct.

Said food additive or food composition comprising said composition maybe solid, semisolid or in a liquid form. Further it may be freeze dried,spray dried or lyophilised. The invented food additive may be used inany kind of food product as well as being used alone. Examples of foodproducts are fat, butter, margarine, oils, cream, milk, cheese, brie,flour, juices, soft drinks, teas. Other examples are yoghurt, ice cream,cakes, bread and dressing.

The invented composition may also be used as a pharmaceuticalcomposition. The pharmaceutical composition comprises the inventedcomposition as well as a pharmaceutically acceptable buffer, excipient,carrier or diluent. Examples of diseases to be treated are the metabolicsyndrome either as a disease or a disorder such as hypertension,arteriosclerosis, gout, diabetes type one and two, cancers anddyslipidemia.

“Pharmaceutically acceptable” means a non-toxic material that does notdecrease the effectiveness of the biological activity of the activeingredients, i.e., the antimicrobial peptide(s). Such pharmaceuticallyacceptable buffers, carriers or excipients are well-known in the art(see Remington's Pharmaceutical Sciences, 18th edition, A. R Gennaro,Ed., Mack Publishing Company (1990) and handbook of PharmaceuticalExcipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000).

The term “buffer” is intended to mean an aqueous solution containing anacid-base mixture with the purpose of stabilising pH. Examples ofbuffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes,HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate,borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate,CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole,imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO andTES.

The term “diluent” is intended to mean an aqueous or non-aqueoussolution with the purpose of diluting the peptide in the pharmaceuticalpreparation. The diluent may be one or more of saline, water,polyethylene glycol, propylene glycol, ethanol or oils (such assafflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The term “adjuvant” is intended to mean any compound added to theformulation to increase the biological effect of the peptide. Theadjuvant may be one or more of zinc, copper or silver salts withdifferent anions, for example, but not limited to fluoride, chloride,bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, carbonate,lactate, glycolate, citrate, borate, tartrate, and acetates of differentacyl composition.

The excipient may be one or more of carbohydrates, polymers, lipids andminerals. Examples of carbohydrates include lactose, sucrose, mannitol,and cyclodextrines, which are added to the composition, e.g., forfacilitating lyophilisation. Examples of polymers are starch, celluloseethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose,hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates,carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid,polysulphonate, polyethylenglycol/polyethylene oxide,polyethyleneoxide/polypropylene oxide copolymers,polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, andpolyvinylpyrrolidone, all of different molecular weight, which are addedto the composition, e.g., for viscosity control, for achievingbioadhesion, or for protecting the lipid from chemical and proteolyticdegradation. Examples of lipids are fatty acids, phospholipids, mono-,di-, and triglycerides, ceramides, sphingolipids and glycolipids, all ofdifferent acyl chain length and saturation, egg lecithin, soy lecithin,hydrogenated egg and soy lecithin, which are added to the compositionfor reasons similar to those for polymers. Examples of minerals aretalc, magnesium oxide, zinc oxide and titanium oxide, which are added tothe composition to obtain benefits such as reduction of liquidaccumulation or advantageous pigment properties.

The compositions of the invention may also be in the form of polymergels, where polymers such as starch, cellulose ethers, cellulosecarboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethylcellulose, ethylhydroxyethyl cellulose, alginates, carageenans,hyaluronic acid and derivatives thereof, polyacrylic acid,polysulphonate, polyethylenglycol/polyethylene oxide,polyethyleneoxide/polypropylene oxide copolymers,polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, andpolyvinylpyrrolidone are used for thickening of the solution containingthe peptide.

The pharmaceutical compositions may be subjected to conventionalpharmaceutical operations such as sterilisation and/or may containconventional adjuvants such as preservatives, stabilisers, wettingagents, emulsifiers, buffers, fillers, etc., e.g., as disclosedelsewhere herein.

Suitable preparation forms are, for example granules, powders, tablets,coated tablets, (micro) capsules, syrups, emulsions, microemulsions,defined as optically isotropic thermodynamically stable systemsconsisting of water, oil and surfactant, liquid crystalline phases,defined as systems characterised by long-range order but short-rangedisorder (examples include lamellar, hexagonal and cubic phases, eitherwater- or oil continuous), or their dispersed counterparts, gels,ointments, dispersions, suspensions, creams, aerosols, droplets orinjectable solution in ampoule form and also preparations withprotracted release of active compounds, in whose preparation excipients,diluents, adjuvants or carriers are customarily used as described above.

The pharmaceutical compositions will be administered to a patient in apharmaceutically effective dose. By “pharmaceutically effective dose” ismeant a dose that is sufficient to produce the desired effects inrelation to the condition for which it is administered. The exact doseis dependent on the, activity of the compound, manner of administration,nature and severity of the disorder, age and body weight of the patientdifferent doses may be needed. The administration of the dose can becarried out both by single administration in the form of an individualdose unit or else several smaller dose units and also by multipleadministration of subdivided doses at specific intervals.

The present invention concerns both humans and other mammal such ashorses, dogs, cats, cows, pigs, camels, among others. Thus the methodsare applicable to both human therapy and veterinary applications. Theobjects, suitable for such a treatment may be identified bywell-established hallmarks.

Here follows, as an example of the invention, a description of theisolation of chloroplast membranes, the thylakoids, from spinach andtheir application in inhibition of the pancreatic lipase activity andreduction of food intake. Following examples are intended to illustrate,but not to limit, the invention in any manner, shape, or form, eitherexplicitly or implicitly.

Example 1 Preparation of Membranes and Membrane Proteins

Thylakoids were isolated as described in Danielsson et al. BiochimBiophys Acta 1608, 53-61 (2004) for use in the lipase assay. Forpreparing food, the thylakoids were isolated as follows: Leaves werehomogenised in a blender and filtered through four layers of nylon mesh(20 μm). The filtrate was centrifuged at 5000 g for 10 min to collectthe thylakoids. These were washed by resuspension in water andrecentrifuged as before. Lipid extraction: 4 ml thylakoid suspension(3.8 mg chlorophyll/mL) mixed with 40 mL chloroform/methanol wasincubated for 1 hr on ice. After centrifugation at 4000 g for 10 min thepellet was extracted for a second time and centrifuged as before. Thepellet was dried in air and extracted with 10 mL of the buffer solutionused for thylakoid isolation on ice to remove water soluble proteins.The mixture was centrifuged at 4000 g for 10 min and the pelletcollected. This is named “membrane protein fraction” (FIG. 1). Trypsintreatment was carried out by incubating the thylakoids with 300 μgtrypsin (Sigma type III)/mg chlorophyll, in 20 mM phosphate buffer (pH7.4), for 45 min at 37° C. After adding 1 mM phenylmethylsulphonylfluoride (PMSF), to inhibit the trypsin, the thylakoids were collectedby centrifugation for 10 min at 900 g. Chlorophyll was determined asdescribed above (Danielsson et al. Biochem Biophys Acta 1608, 53-61(2004) and protein according to Bradford (Bradford, M. M. Anal Biochem72, 248-54 (1976)). Light harvesting complex II (LHCII) was prepared asdescribed in Andersson, B. & Albertsson, P.-Å. J. Chromatogr. 890,131-141 (1981). Mitochondria prepared from potato tubers according to(Petit, P. X., Edman, P., Gardestrom, P. & Ericson, I. Biochim BiophysActa 890, 377-386 (1987)). Plasma membranes from spinach leaves preparedaccording to (Kjellbom, P. & Larsson, C. Physiol Plant 62, 501-509(1984)). Membranes from Synechosystis prepared according to (Norling,B., Zak, E., Andersson, B. & Pakrasi, H. FEBS Lett 436, 189-92(1998))-Chromatophores from Rhodospirillum rubrum prepared according to(Wang, H., Franke, C. C., Nordlund, S. & Noren, A. FEMS Microbiol Lett253: 273-279 (2005)). Before use, extrinsic water soluble proteins wereremoved by washing with 0.5 M NaCl, 25 mM Tris-HCl, pH 7.8 followed bytwo washings with the Tris buffer only according to (Wang, H., Franke,C. C., Nordlund, S. & Noren, A. FEMS Microbiol Lett 253: 273-279(2005)). Transhydrogenase prepared according to (Althage, M. et al.Biochim Biophys Acta 1659, 73-82 (2004)) from E. coli. Cytochrome b₆ffrom spinach leaves were prepared according to (Romanowska, E. &Albertsson, P.-Å. Plant Cell Physiol 35, 557-568 (1994)).

Example 2 Crude Preparation of Cell Membranes (Thylakoids)

Leaves of spinach, either fresh or frozen, were cut into pieces. Thesewere suspended in water and disintegrated by a mechanical mincer untilmost of the cells are broken. The slurry was then filtered through anylon net with a pore size of 20 μm. The filtrate is centrifuged at2000×g for 5 min. The pellet was resuspended in water andrecentrifugated at 2000×g for 5 min. The pellet was stored frozen ordried. Alternatively, a precipitating agent was added to the filtrate:The filtrate was acidified by addition of acid to a low pH, such as pH4-5, so that the membranes precipitate. The precipitate was washed byresuspension in water at pH 4-5 and resettling of the precipitate. Theprecipitate is collected.

Example 3 Preparation of Crude Cell Membrane Fractions

Cells, suspended in water, were further disintegrated mechanically by amincer until most of the cells are broken. The slurry was filtrated toremove unbroken cells and large fragments of cell walls. The filtratewas centrifuged at 10 000×g for 10 min. The pellet was resuspended andrecentrifuged at 10 000×g for 10 min. The pellet was collected andstored frozen or dried.

Example 4 Preparation of Thylakoids from Spinach Using Filtration Only

Leaves of spinach, either fresh or frozen, were cut into pieces. Thesewere suspended in water and disintegrated by a mechanical mincer untilmost of the cells are broken. The slurry was then filtered through anylon net with a pore size of 20 μm. To the filtrate is added aceticacid until the pH reaches 4.7 to induce flocculation. When theflocculate has settled in the bottom of the vessel the supernatant isremoved by decanting. The flocculate is then put on a filter with a poresize of 20 μm. The flocculated thylakoids stay at the filter and can bewashed with water at pH 4.7. The washed thylakoids are collected andafter adjusting to a desired pH they are stored frozen or dried.

Example 5 Measurement of the Lipase Activity

The lipase activity was measured according to the following (BorgstromB. and Erlanson C. European J. of Biochemistry 37: 60-68, 1973): 15 mlof an aqueous solution containing 1 mM Tris buffer pH 7.0, 1 mM calciumchloride, 150 mM sodium chloride, 4 mM sodiumtaurodeoxycholate, 0.5 μglipase, 1 μg of colipase.

To this mixture 0.5 ml of tributyrin is added together with increasingamounts of membranes and the released fatty acids measured using a pHstat.

The effect of the biological membranes on lipase activity is shown inFIG. 1-2. The effect of membrane proteins and synthesized polypeptide isfurther shown in FIG. 3.

Example 6 Production of a Food Product and the Effect on Food Intake

Cakes (500 g, 42% fat by energy) were prepared in the following way:

Ingredient Quantity Gelatine 20 g (dissolved in 200 ml water and heatedin water-bath to 60-70° C.) Casein 110 g Starch 190 g Corn-oil 15 g Lard90 g Mix of vitamin 5 g Salt mix 20 g Choline 1 g Methionine 1.5 gCellulose 47.5 g

Chloroplast membranes (Thylakoids, containing 1000 mg chlorophyll)suspended in 30 g water

Procedure.

Starch and oil was mixed in a food processor (Bosch Universal). Theremaining solid ingredients were added, followed by the gelatinesolution. After thoroughly mixing, water, with or without chloroplastmembranes (thylakoids), was finally added. The cakes were baked in anoven at 70° C. for two days.

The experiment shown in FIG. 4 was performed in the following way.

Female Sprague-Dawley rats (200 g) from B&K, Sollentuna, Sweden werehoused in a temperature-controlled room (22±1° C.) under a 12-h light(6:00-18:00)/12-h cycle, given free access to water, and fed ad libitumon a standard chow unless otherwise stated during high-fat dietexperiments. All procedures using animals were approved by the LocalAnimal Ethics Committee Lund, Lund, Sweden.

Feeding Protocol

For measurement of food consumption rats were individually housed incage and given a high-fat diet for one week before the start of thestudy. The high-fat diet consisted of a diet, containing by energy 42.1%fat, 23.9% protein and 34.0% carbohydrate with a caloric density of 4.7kcal/g as described (Lindquist et al. Regul. Pept. 130: 123-132 (2005).The high-fat diet containing thylakoids were prepared as for thehigh-fat diet with the addition of purified thylakoids at aconcentration of 2 mg chlorophyll per gram of food. Food intake wasmeasured daily and body weight at the start and end of the feedingperiod. Cages were carefully monitored for evidence of food spillage.

TABLE 1 Effect of treatment with thylakoids during high- fat diet foreleven days in Sprague-Dawley rat, see FIG. 4. Control Thylakoids Bodyweight gain (g) 60.5 ± 3.55 49.9 ± 8.62* Serum TG (mmol/L) 1.02 ± 0.130.62 ± 0.04* Plasma CCK (pmol/L) 0.68 ± 0.08 0.86 ± 0.12* Values aremeans ± SE with *Significance level of P < 0.05 and **significance levelof P < 0.01 between control diet and thylakoid treatment. TG =triglycerides, CCK = cholecystokinin

Example 7 Food Emulsion

The vegetables (300 g) were allowed to thaw at room temperature for 0.5hours before they were thoroughly processes in a household mixer.Vegetable juice was obtained by feeding the finely cut pieces ofvegetable into a household juice centrifuge.

A smooth well-tasting emulsion was obtained by homogenizing thevegetable juice (60 g) and triglyceride oil (30 g) with a handheldsmixer equipped with stainless steal cutting blades. Emulsions made frombroccoli and spinach juice were visually inspected for free oil and/orwater phase after 0.5 hours (storage at room temperature) and 10 hours(storage in refrigerator).

Appearance after 0.5 Appearance after 10 Vegetable hour of storage. hourof storage. Spinach No free oil phase, no free No free oil phase, nowater phase free water phase Broccoli No free oil phase, no free No freeoil phase, free water phase water phase

The results from the inspection are summarised in table above and showthat the oil droplets in the emulsions are very stable since nocoalesces could be notice (no free oil phase).

The invention claimed is:
 1. A method of suppressing appetite in amammal comprising administering to a mammal in need thereof an effectiveamount of a composition comprising an isolated thylakoid membrane or aportion thereof to suppress appetite.
 2. The method according to claim1, wherein the thylakoid membrane or portion thereof, is obtained from aplant, algae or microorganism, or is a mixture thereof.
 3. The methodaccording to claim 2, wherein the thylakoid membrane or portion thereofis obtained from clover, rape, sugar beet, dandelion, Arabidopsisthaliana, maize, tobacco, sunflower, Chenopodium, Atriplex, spinach,grasses, or a mixture thereof.
 4. The method according to claim 3,wherein the thylakoid membrane or portion thereof is obtained fromspinach.
 5. The method according to claim 1, wherein the thylakoidmembrane or portion thereof comprises hydrophobic amino acid residues.6. The method according to claim 1, wherein said composition furthercomprises at least one hydrophobic peptide having from about 15 to about25 amino acid residues.
 7. The method according to claim 1, wherein thecomposition further comprises two or more hydrophobic peptides beingderived from the same protein or different proteins.
 8. The methodaccording to claim 1, wherein said composition further compriseshydrophobic peptides comprising 16, 17, 18, 19, 20, 21, 22, 23 or 24amino acid residues.
 9. The method according to claim 1, wherein saidcomposition further comprises hydrophobic peptides comprising SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, or a mixture thereof.
 10. The methodaccording to claim 1, wherein the thylakoid membrane or portion thereofhas a size distribution of 0.1 μm to about 5 μm.
 11. The methodaccording to claim 1, wherein said composition further compriseshydrophobic peptides comprising from about 1 to about 50 additionalamino acid residues.
 12. The method according to claim 1, wherein saidcomposition further comprises hydrophobic peptides that have beenmodified by amidation, esterification, acylation, acetylation,PEGylation or alkylation.
 13. The method according to claim 1, whereinsaid composition further comprises a pharmaceutically acceptablecarrier, diluent, excipient or buffer.
 14. The method according to claim1, wherein said composition is a food composition.
 15. The methodaccording to claim 14, wherein said food composition is margarine, oil,cream, milk, cheese, brie, flour, juice, soft drink or tea product. 16.The method according to claim 1, wherein said composition is freezedried, spray dried or lyophilised.
 17. The method of claim 1, whereinthe composition regulates the appetite of the mammal being treated. 18.The method according to claim 1, wherein said composition furthercomprises hydrophobic peptides comprising SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, or a mixture thereof.
 19. The method according to claim 1,wherein the composition reduces lipolytic activity, retards fatdigestion, regulates appetite, or lowers blood lipids in the mammalbeing treated.